EP1939212A1 - Organische Verbindungen - Google Patents

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Publication number
EP1939212A1
EP1939212A1 EP06126727A EP06126727A EP1939212A1 EP 1939212 A1 EP1939212 A1 EP 1939212A1 EP 06126727 A EP06126727 A EP 06126727A EP 06126727 A EP06126727 A EP 06126727A EP 1939212 A1 EP1939212 A1 EP 1939212A1
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EP
European Patent Office
Prior art keywords
extraction
interest
polypeptide
process according
cell
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EP06126727A
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English (en)
French (fr)
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Lek Pharmaceuticals dd
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Lek Pharmaceuticals dd
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Priority to EP06126727A priority Critical patent/EP1939212A1/de
Priority to PCT/EP2007/063900 priority patent/WO2008074724A1/en
Publication of EP1939212A1 publication Critical patent/EP1939212A1/de
Ceased legal-status Critical Current

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14Extraction; Separation; Purification
    • C07K1/36Extraction; Separation; Purification by a combination of two or more processes of different types
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/53Colony-stimulating factor [CSF]
    • C07K14/535Granulocyte CSF; Granulocyte-macrophage CSF
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00Preparation of peptides or proteins
    • C12P21/02Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a new process for the production of a polypeptide of interest, or of a precursor thereof, wherein the protein or the precursor are expressed in a host cell comprising a cell wall and at least one cell membrane, followed by extraction from the intact host cell by partial permeabilization of the cell membrane under non-denaturing conditions. During extraction the polypeptide of interest, or the precursor thereof, which is expressed in at least partially correctly folded form, passes through the cell wall and the cell membrane.
  • the principle of the present invention can be applied to a wide range of heterologous and homologous proteins as well as fusion proteins expressed in a variety of host cells, e.g. in E. coli. Exemplarily the process is described by referring to the process for the production of Green Fluorescent Protein (GFP) and human Granulocyte-Colony Stimulating Factor (hG-CSF).
  • GFP Green Fluorescent Protein
  • hG-CSF human Granulocyte-Colony Stimulating Factor
  • the isolation of the expressed protein from a host cell is a crucial step in the production of proteins.
  • the polypeptide of interest can be accumulated within the cell in different forms.
  • a polypeptide of interest may be accumulated in soluble form within the cytoplasm of the host cell, it may be transported into the periplasmic space, or it may be accumulated inside of the cell in so called "inclusion bodies".
  • export of the polypeptide of interest into the bacterial periplasm can be induced with the aid of a pro- or eukaryotic signal sequence. Because of the low transport capacity of the bacterial export machinery usually only very small quantities of product can be accumulated in this way.
  • the way of deposition of the protein within the host cell determines how it can be retrieved and which further processing is required before the protein can be subjected to purification.
  • the extraction of proteins of interest from host cells is generally started by disruption of the host cell. This can be achieved e.g. by mechanical disruption or by enzymatic lysis of the cells.
  • Classical mechanical disruption requires the use of high-pressure homogenizers.
  • Disruption must be performed in a closed system to avoid the release of spray which contains bacterial endotoxins into the surrounding.
  • enzymatic cell disruption e.g. by lysozyme
  • the cell membranes and the cell walls of the cells are demolished by enzymatic lysis. This way, a comparable result as with the mechanical cell disruption is achieved.
  • the remaining cell fragments must be separated from the liquid phase of the suspension. This is generally achieved by centrifugation, which has to be performed in a closed system as well, due to the spray of bacterial endotoxins.
  • Proteins which are expressed soluble within the cell are generally separated from the cell debris by microfiltration or centrifugation.
  • the resulting protein solution then needs to be subjected to further purification steps, which usually include a sequence of chromatographic purification steps.
  • polypeptide of interest is over expressed in bacterial cells, especially when strong promoters are used, often high density polypeptide aggregates are formed, which are deposited in the cytoplasm of the cell as so called inclusion bodies.
  • the production of polypeptides in the form of inclusion bodies is of special interest for production on industrial scale, since the expressed polypeptide is present in high amounts in the inclusion bodies.
  • proteins deposited in inclusion bodies are protected from cellular proteases.
  • Such work up usually involves e.g. solubilization of the inclusion bodies using high concentration of denaturants, followed by a renaturation step.
  • the preparation of proteins from inclusion bodies starts with a washing step.
  • the washing solution usually contains a detergent, e.g. Deoxycholate, or Triton X100. Washing is followed by solubilization of the inclusion bodies. Due to the low solubility of the inclusion bodies this step generally requires the use of strong denaturants, e.g. high concentrations of urea or guanidium chloride. Solubilization of the inclusion bodies is followed by in vitro renaturation. This step requires large process volumes. Thus processing of inclusion bodies is complex, especially since correct refolding is required during the renaturation step in order to gain the biologically active form of the polypeptide of interest.
  • the present invention it was surprisingly found that it is possible to extract a polypeptide of interest from inside the host cell without cell disruption if, for a host cell which comprises a cell wall in addition to at least one cell membrane, the cell membrane is partially permeabilized in such a way, that certain molecules can diffuse through the partially permeabilized membranes, while the mechanical stability of the host cell remains intact. This is achieved by incubating the host cell in mild detergents under non-denaturing conditions. Following the partial permeabilization the polypeptide of interest is extracted from the cell and accumulated in the liquid part of the cell suspension.
  • the process of the present invention provides a more selective way of extraction of the polypeptide of interest, during which also less host cell DNA is released from the cell.
  • the cells are not fragmented and can easily be separated from the protein solution either by centrifugation or by expanded bed adsorption techniques. If the polypeptide of interest is expressed by the host cell in form of classical or non-classical inclusion bodies, in addition to cell disruption also the steps of de- and renaturation become unnecessary. In this case moreover, since the extraction is carried out under non-denaturing conditions, after extraction the polypeptide of interest is soluble and in certain cases also biologically active.
  • the present invention relates to a new process for the production of a heterologous or homologous polypeptide of interest, wherein the polypeptide of interest or a precursor thereof is expressed in at least partially correctly folded form within a host cell, which comprises a cell wall and at least one cell membrane.
  • a host cell which comprises a cell wall and at least one cell membrane.
  • the polypeptide of interest is extracted from said host cell by partially permeabilizing the cell membrane under non-denaturing conditions using mild detergents. This partial permeabilization of the cell membrane allows diffusion of certain molecules e.g. the polypeptide of interest, through the cell membrane, while the cell, supported by its cell wall, remains intact.
  • the polypeptide of interest is accumulated in the liquid part of the cell suspension and can easily be separated from the intact cell, e.g. either by centrifugation or expanded bed adsorption techniques.
  • the present invention is related to a process for the recombinant production of a polypeptide of interest within a host cell which comprises a cell wall and at least one cell membrane, comprising:
  • a heterologous or homologous polypeptide of interest or a precursor of said protein, expressed within a host cell which comprises a cell wall and at least one cell membrane in at least partially correctly folded form (in soluble form or in form of classical or non-classical inclusion bodies) can be isolated from the host cell by the use of mild detergents under non-denaturing conditions without prior cell disruption.
  • the present invention preferably refers to a process, wherein the extraction of the polypeptide of interest is performed using a mild detergent.
  • the present invention refers to a process, wherein the extraction of the polypeptide of interest is performed using NLS.
  • the extraction according to the present invention is performed using NLS in a concentration of between 0.05% and 0.2%.
  • the present invention refers also to the use of NLS in the extraction according to the present invention.
  • detergent refers to any ionic or non-ionic substance or mixtures of such substances able to form micelles.
  • wash detergent refers to a detergent which, while enabling permeabilization of the host cell membranes, keeps the cell intact without affecting the biological activity of the polypeptide of interest.
  • suitable concentration for a particular detergent is determined experimentally. A person skilled in the art can determine the suitable concentration without undue burden.
  • intact cell refers to a cell that is mechanically intact, not disrupted, nor collapsed or burst.
  • the suitable concentration of a particular detergent can be determined by incubation of the pure biologically active polypeptide of interest in solutions comprising various concentrations of that detergent under the same conditions (e.g. temperature, buffering system, pH) that are chosen for the extraction.
  • the biological activity of the polypeptide of interest is assessed for each solution comprising different concentrations of said detergent.
  • Tests for biological activity of proteins are generally known. One such method is described in example 8.
  • denaturation refers to a process in which the native conformation of the protein (three dimensional structure) is changed, but the primary structure (amino acid chain, peptide links) of the protein remains unchanged. Upon denaturation the protein looses its biological activity.
  • renaturation refers to a process in which, starting from a denatured protein, the native conformation of the protein (three dimensional structure) is restored. Upon renaturation the protein regains its biological activity.
  • non-denaturing conditions refers to conditions, in which the native conformation of the protein, its three dimensional structure, is maintained. The biological activity of a protein is preserved under such conditions.
  • partial permeabilization refers to the removal of some membrane proteins from the host cell.
  • certain molecules can pass through the cell membrane, while the cell remains intact.
  • E.g. water, ions, extraction substances can pass from the outside into the cell, while molecules present inside of the cell, e.g. proteins, can pass in the opposite direction.
  • heterologous protein refers to a protein which is foreign to the host organism in which it is expressed.
  • homologous refers to a protein which is naturally expressed by the host organism.
  • polypeptide of interest is expressed soluble in the cytoplasm of the host cell.
  • polypeptide of interest is expressed within the host cell in the form of non-classical inclusion bodies.
  • polypeptide of interest is expressed within the host cell in the form of classical inclusion bodies.
  • inclusion bodies is generally understood to denote compact aggregates of incorrectly folded or partially correctly folded proteins which are highly insoluble so that strong denaturants are needed for solubilization. These inclusion bodies are referred to as “classical inclusion bodies” in the following.
  • non-classical inclusion bodies refers to inclusion bodies which are characterized by their greater solubility and higher content of correctly folded protein and/or correctly folded precursor of the polypeptide of interest, when compared to classical inclusion bodies. These non-classical inclusion bodies are soluble under non-denaturing conditions, e.g. in non-denaturing aqueous solutions. Non-classical inclusion bodies are formed under certain specific fermentation conditions, as described in S. Jevsevar et al. (2005), Biotechnolog. Prog. 21, 632-639 which is herewith incorporated by reference.
  • the term "precursor" of the polypeptide of interest as used herein refers to a protein (e.g. G-CSF) having the correct conformation (the native conformation may be present or inherently generated), wherein the disulfide bridges may not be formed or not yet be formed completely. After extraction of the precursor from the host cell the intramolecular disulfide bridges form either spontaneously or their formation is induced by addition of a catalyst, such as a metallic salt, e.g. CuSO 4 , to the washing solution.
  • a catalyst such as a metallic salt, e.g. CuSO 4
  • the process of the present invention can be used for proteins expressed in any host organism, provided that it has a cell wall and at least one cell membrane.
  • Suitable host organisms can be e.g. bacteria or yeasts. From suitable bacterial hosts E. coli and Streptomyces sp., Bacillus sp. are preferred, E. coli is a particularly preferred host organism. Of potential yeast host organisms e.g. Saccharomyces cerevisiae or Pichia pastoris, Hansenula polymorpha, or Candida utilis can be employed.
  • the present invention relates to a process for the production of a polypeptide of interest, wherein the polypeptide of interest is expressed in a bacterial or a yeast host cell.
  • the polypeptide of interest is expressed in a bacterial host cell.
  • Suitable host cells are transformed with plasmids in a manner known per se, in order to express the respective polypeptide of interest. Fermentation conditions known in the art are chosen and the process is adjusted in such a way, that the respective polypeptide of interest or a precursor thereof is expressed in an at least partially correctly folded form. Preferably fermentation conditions are chosen, that result in protein expression soluble within the cytoplasm of the cell, or in form of classical or non-classical inclusion bodies.
  • the cells are centrifuged in a manner known in the art and the supernatant is discarded.
  • the cell pellet is re-suspended in extraction solution.
  • the cells may be incubated in extraction solution for a certain period. Following this incubation the cells are centrifuged and the supernatant is collected. This sequence of steps is referred to in the following as an "extraction cycle". The remaining cell pellet may be re-suspended in fresh extraction solution, while the supernatant can be collected for further processing. The whole extraction can be repeated in identical cycles as described above, while the supernatant is collected after each incubation cycle.
  • a single extraction cycle may be sufficient for extraction of the polypeptide of interest.
  • the extraction according to the present invention is performed by a single extraction cycle or, equally preferably, by a series of consecutive extraction cycles.
  • the extraction is performed by a series of between 2 and 4 consecutive extraction cycles.
  • the duration of the extraction cycles may be between 1 and 20 hours, preferably it is between 3 and 5 or, equally preferably, between 8 and 10 hours.
  • the extraction may also be performed as a two-step procedure, wherein in a first step the cells are incubated in a permeabilization solution followed by extraction in a second step as described above. Also after partial permeabilization extraction can be performed either by a single extraction cycle or by repeated cycles, as described above. According to the present invention prior to extraction the host cell may be partially permeabilized by incubation in a solution comprising a mild detergent, e.g. NLS (N-lauroyl sarcosine).
  • a mild detergent e.g. NLS (N-lauroyl sarcosine).
  • the host cell prior to extraction, is subjected to a partial permeabilization step.
  • the host cell is partially permeabilized by incubation in a solution comprising a mild detergent.
  • the mild detergent is NLS.
  • the concentration of NLS for partial permeabilization is between 0.05% and 0.2%. Most preferably the concentration of NLS is 0.1 %.
  • the present invention provides a process for the extraction of a biologically active polypeptide of interest from classical or non-classical inclusion bodies, while these inclusion bodies are still inside the cell.
  • extraction substances can also be used for the extraction of proteins of interest which are present in the host cell in soluble form.
  • extraction substance refers to water soluble, small, polar molecules, which do not permeabilize the cell-membranes but enhance the dissolution of a polypeptide of interest which is deposited within the cell e.g. in form of classical or non-classical inclusion bodies or in soluble form, under non-denaturing conditions.
  • the extraction of the polypeptide of interest is performed using an extraction solution comprising a mild detergent and in addition an extraction substance.
  • NLS is used in a concentration of between 0.05% and 0.1 %
  • NDSB is used in a concentration of between 0.2% and 2%.
  • NLS is used in a concentration of 0.1 %
  • NDSB is used in a concentration of 2%.
  • extraction solutions comprising only a mild detergent (e.g. NLS) and extraction solutions comprising an extraction substance (e.g. NDSB) in addition to a mild detergent (e.g. NLS) are applied in alternating extraction cycles.
  • a mild detergent e.g. NLS
  • extraction solutions comprising an extraction substance (e.g. NDSB) in addition to a mild detergent (e.g. NLS) are applied in alternating extraction cycles.
  • the partial permeabilization is performed for about 30 minutes in a solution comprising about 0,05% - 0,2%NLS, while the subsequent extraction steps are performed in an extraction solution comprising about 0,2-2% NDSB.
  • extraction may be performed in an extraction solution comprising only an extraction substance, and no mild detergent.
  • the extraction from a partially permeabilized host cell is performed using an extraction solution comprising an extraction substance and no mild detergent.
  • the extraction solution comprises NDSB and no mild detergent.
  • the extraction solution comprises NDSB in a concentration between 0.2% and 2% and no mild detergent.
  • an extraction solution comprising an extraction substance (e.g. NDSB) in addition to a mild detergent (e.g. NLS) may be used for the extraction of proteins that are expressed in form of classical or non-classical inclusion bodies, or soluble within the cell, e.g. G-CSF, GFP or IFN-alpha.
  • an extraction substance e.g. NDSB
  • a mild detergent e.g. NLS
  • an extraction solution comprising only an extraction substance (e.g. NDSB) and no mild detergent may be used for the extraction of proteins that are expressed in form of classical or non-classical inclusion bodies, or soluble within the cell, e.g. G-CSF, GFP or IFN-alpha.
  • an extraction substance e.g. NDSB
  • no mild detergent may be used for the extraction of proteins that are expressed in form of classical or non-classical inclusion bodies, or soluble within the cell, e.g. G-CSF, GFP or IFN-alpha.
  • an extraction solution comprising only a mild detergent (e.g. NLS) and no extraction substance may be used for the extraction of proteins that are expressed in form of non-classical inclusion bodies or soluble within the cell.
  • This process can be employed e.g. for the extraction of G-CSF, GFP or IFN-alpha.
  • polypeptide of interest is G-CSF.
  • the present invention further relates to the purification of a protein, produced by the process according to the present invention.
  • the present invention also refers to the use of NLS as mild detergent for the partial permeabilization of a cell membrane of a host cell within the process of the present invention.
  • the present invention refers as well to the use of NDSB as an extraction substance within the process of the present invention.
  • All extraction cycles may be run at a temperature of about 2°C to about 25°C, more preferably at a temperature between about 4°C and 20 °C, 4°C being most preferred.
  • the pH of the extraction solution is adjusted depending on the respective protein to be extracted.
  • the aim is to maximally stabilize the polypeptide of interest. It is known to a person skilled in the art how to select, adjust and maintain the optimal pH for each polypeptide of interest.
  • the solution comprising the extraction substance may be buffered, but depending on the stability of the respective polypeptide of interest un-buffered solutions can also be applied.
  • the process disclosed in the present invention results in a cell suspension comprising the extraction solution, the intact host cells and the polypeptide of interest or its precursor.
  • This cell suspension can directly be subjected to the first step in downstream processing, e.g. expanded bed adsorption (EBA).
  • EBA expanded bed adsorption
  • the cells can be removed from the protein solution by centrifugation and the solution can then be applied to further purification steps, e.g. to a first chromatographic step (capture phase).
  • the extraction procedure can be coupled with expanded bed adsorption (EBA).
  • EBA expanded bed adsorption
  • the process of the present invention can be employed for the production of a wide range of pharmaceutically active proteins, e.g. for the production of interferons (IFN), such as IFN-alpha, IFN-beta, IFN gamma, interleukins (IL), such as IL-2 and IL-4, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage stimulating factor, (M-CSF), epidermal growth factor (EGF), human serum albumin (HAS), deoxyribonuclease (DNAse), fibroblast growth factor (FGF); tumor necrosis factor alpha (TNF alpha) and tumor necrosis factor beta (TNF beta), fragments of antibodies, e.g., Fab fragments.
  • proteins that can be produced by the process of the present invention comprise proteins without pharmaceutical activity e.g. GFP, and fusion proteins of either pharmaceutically active or pharmaceutically non-active proteins.
  • compositions of buffers used in examples are provided.
  • Biosynthesis is performed in a 5 litre laboratory fermentor at 25-30° C using the production strain E. coli BL21 (DE3)-pET9a-Fopt5. Most of G-CSF is expressed in form of non-classical inclusion bodies. The host cells are centrifuged and the supernatant is discarded.
  • Example 2 Two step extraction of G-CSF from partially permeabilized E. coli cells usin g NLS
  • step 1 the wet bacterial pellet is re-suspended in 5 volumes of a permeabilization buffer (PB1 and aliquoted (approximately 1 gram of wet pellet per aliquot). After 30 minutes of incubation (permeabilization) the suspension is again centrifuged. The supernatant is discarded and the remaining pellet frozen at -20°C.
  • step 2 based on the thus prepared aliquots, the protein extraction is performed with either extraction buffer EB1 or EB2, at either +4°C or +20°C resulting in four different experimental settings (see Table 1). Within each experimental setting four consecutive extractions are performed. All extractions are run for approx. 8-10 hours and are performed in glass vials under gentle stirring. Table 1: 1.
  • experimental setting 1 extraction with 10 volumes of EB1 at + 4° C 2.
  • experimental setting 2 extraction with 10 volumes of EB1 at + 20° C 3.
  • experimental setting 3 extraction with 10 volumes of EB2 at + 4° C 4.
  • experimental setting 4 extraction with 10 volumes of EB2 at + 20° C
  • Example 3 Two step extraction of G-CSF from partially permeabilized E. coli cells using NDSB
  • G-CSF is expressed as described in example 1.
  • a two-step extraction is applied as in example 2.
  • cells are permeabilized with 10 volumes of PB2 for approximately 30 minutes.
  • step 2 all subsequent extractions are performed with 10 volumes of EB3 or EB4 both at +4°C. All extractions are run for approx. 8-10 hours and are performed in glass vials under gentle stirring.
  • G-CSF is expressed as described in example 1. Then 10 volumes of 0.2%NLS or 0.1%NLS are used at +4°C in step 1, for partial permeabilization, combined with only one extraction in step 2, of approximately 8-10 hours duration, with 25 volumes of EB3.
  • G-CSF is expressed as described in example 1.10 volumes of 0.05%NLS are used in step 1 for permeabilization (permeabilization is performed as described in example 2) and 20 volumes of 2.0%NDSB-201 in 40mM TRIS/HCl pH 8.0 containing 0.05% NLS in step 2 for extraction. Again extraction is performed as described in example 2
  • Example 4 Two step extraction of G-CSF from partially permeabilized E. coli cells using a combination of NLS and NDSB
  • G-CSF is expressed as described in example 1.
  • Host cells are re-suspended in 10 volumes of permeabilization buffer PB3, e.g., 1g wet pellet in 10ml of buffer. After 40 min incubation at +4°C using gentle mixing, the suspension of cells is centrifuged for 10 minutes at +4°C, 5000 rpm and the pellet is re-suspended in 20 volumes of the extraction buffer EB5. The cells are suspended over night in extraction solution at +4°C under gentle mixing.
  • Example 5 One step extraction of G-CSF using NLS or a combination of NLS and NDSB
  • G-CSF is expressed as described in example 1. Host cells (1g wet pellet) are re-suspended in minimum 10 ml of extraction solution EB1 or EB5 and extracted over night at +4°C under gentle mixing.
  • GFP GFP protein kinase
  • GFP is expressed soluble within the host cell. While omitting the permeabilization step, extraction is performed as described in example 2.
  • the amount of total G-CSF in the pooled extract is determined densitometrically on commercially purchased SDS-PAGE gels (NuPage 4-12 % Bis-TrisGel, Invitrogen (catalogue 2003, no.NP0321BOX), in the presence of a reducing agent using the NuPage electrophoresis system.
  • SDS-PAGE gels are Coomassie stained in a generally known manner.
  • G-CSF The biological activity of G-CSF is determined on the NFS-60 cell line according to a published procedure ( Hammerling U. et al. J. Pharm Biomed Anal 13, 9-20 (1995) ) using international standard human recombinant G-CSF 88/502, NIBSC Potters Bar, Hertfordshire, UK.
EP06126727A 2006-12-20 2006-12-20 Organische Verbindungen Ceased EP1939212A1 (de)

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EP06126727A EP1939212A1 (de) 2006-12-20 2006-12-20 Organische Verbindungen
PCT/EP2007/063900 WO2008074724A1 (en) 2006-12-20 2007-12-13 Organic compounds

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WO2011109556A3 (en) * 2010-03-04 2012-01-12 Pfenex Inc. Method for producing soluble recombinant interferon protein without denaturing
CN102906108A (zh) * 2010-03-04 2013-01-30 菲尼克斯公司 用于无变性制备可溶性重组干扰素蛋白的方法
US9187543B2 (en) 2010-03-04 2015-11-17 Pfenex Inc. Method for producing soluble recombinant interferon protein without denaturing
CN102906108B (zh) * 2010-03-04 2016-01-20 菲尼克斯公司 用于无变性制备可溶性重组干扰素蛋白的方法
RU2573909C2 (ru) * 2010-03-04 2016-01-27 Пфенекс Инк. Способ получения растворимого рекомбинантного белка интерферона без денатурирования
US9611499B2 (en) 2010-03-04 2017-04-04 Pfenex Inc. Method for producing soluble recombinant interferon protein without denaturing
KR101857825B1 (ko) 2010-03-04 2018-05-14 피페넥스 인크. 변성 없이 가용성 재조합 인터페론 단백질을 제조하는 방법

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